We investigated oxygen and site-specific nitrogen isotope effects of N2O produced in the NO3−-to-N2O step of denitrification. Arable sand and silt loam soils with varying NO3− availability were incubated under N2/C2H2 atmosphere in order to establish anaerobic conditions and to block N2O reduction. Dual isotope and isotopomer ratios of emitted N2O were determined by analysis of δ18O, average δ15N (δ15Nbulk) and 15N site preference (SP is equal to difference in δ15N between the central and peripheral N positions of the asymmetric N2O molecule). The average N enrichment factor of the NO3−-to-N2O step ranged from −47.9 to −53.6‰, which is between the reported ranges of enrichment factors of nitrification and denitrification. SP varied with time, and mean values were between 3.1 and 8.9‰, which is higher compared to SP reported from pure cultures of denitrifiers but lower compared to nitrifiers and fungal denitrifiers. This shows that SP of N2O produced in soils might be seen as a semiquantitative indicator for the different pathways of N2O production but not specific enough to quantify the relative contribution of denitrification to the total N2O flux in systems where several source processes are important. δ18O of N2O from all treatments was less variable compared to δ18O of soil NO3−, indicating that there was a relatively large O exchange with water during N2O formation.